K562 is an erythroleukemic cell line used as a model for the study of the control of human globin gene expression. These cells do not support transcription of the beta-globin gene but do express transcripts of epsilon- and gamma-globin genes at very high levels when exposed to a number of inducing agents. Results from this and other laboratories suggest that the control of this pattern of expression is mediated by the presence and/or absence of trans-acting factors which exert their action on sequences corresponding to the promoters of these genes. Sequence specific DNA binding protein acting on cis-regulatory elements have been hypothesized to be key elements in eukaryotic gene transcription, and even though considerable progress has been made in their isolation, only one DNA binding proteins with affinity for the human globin gene promoters have been identified. We have defined several positive and negative regulatory regions 5' to the epsilon-globin promoter, and detected binding of proteins to these regions. We have previously reported the presence of a transcriptional control element with properties of a silencer extending from -392 to -177 bp relative to the cap site of the human e-globin gene. We also showed that this silencer has stronger inhibitory activity in HeLa cells than K562 human erythroleukemia cells. Using deletion mutants and synthetic oligonucleotides in transient expression assays, DNA sequences responsible for this effect have been further delimited to 44 nucleotides located between -294 and -251 bp. Gel electrophoresis mobility shift assays and DNasel footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. The protein present in K562 cells, but not in HeLa cells, that interacts specifically with this silencer binds to the same sequence recognized by the yeast silencer binding protein ABF1. Possible mechanisms by which these proteins may regulate epsilon-globin gene transcription in erythroid and non-erythroid cells are discussed.